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Asthma continues to cause morbidity and mortality despite advances in treatment that include biologics targeting Type 2 inflammation. The International Collaborative Asthma Network (ICAN) forum was developed with the primary goal of promoting innovative, collaborative research that focuses on mechanisms and treatment for asthma that does not respond or that responds poorly to currently available treatments. The mission of ICAN is innovation, collaboration, translation, and increasing high quality research. At the second ICAN meeting, presenters covered a broad scope and depth of asthma-related topics in the categories of complex data, novel therapeutics and diagnostics, breath analysis and microbiome, disease mechanisms, systemic effects, and circadian rhythm. Key actionable needs and research topics were identified during the group discussions. The presentations and discussions that occurred at the second ICAN had an immediate impact on asthma research in the form of new collaborations and implementation of new research ideas and techniques. The forum also served to connect early-stage investigators with investigators who are well established, thereby fostering innovation, translation, and collaboration well into the future. A third ICAN meeting is planned for 2025 to further the innovations and collaborations that will translate into novel therapies and diagnostics to improve the lives of patients with asthma.

Background: Asthma pathophysiology is associated with mitochondrial dysfunction. Mitochondrial DNA copy number (mtDNA-CN) has been used as a proxy of mitochondrial function, with lower levels indicating mitochondrial dysfunction in population studies of cardiovascular diseases and cancers., Objectives: We investigated whether lower levels of mtDNA-CN are associated with asthma diagnosis, severity, and exacerbations., Methods: mtDNA-CN is evaluated in blood from 2 cohorts: UK Biobank (UKB) (asthma, n = 39,147; no asthma, n = 302,302) and Severe Asthma Research Program (SARP) (asthma, n = 1283; nonsevere asthma, n = 703)., Results: Individuals with asthma have lower mtDNA-CN compared to individuals without asthma in UKB (beta, -0.006 [95% confidence interval, -0.008 to -0.003], P = 6.23 × 10 -6 ). Lower mtDNA-CN is associated with asthma prevalence, but not severity in UKB or SARP. mtDNA-CN declines with age but is lower in individuals with asthma than in individuals without asthma at all ages. In a 1-year longitudinal study in SARP, mtDNA-CN was associated with risk of exacerbation; those with highest mtDNA-CN had the lowest risk of exacerbation (odds ratio 0.333 [95% confidence interval, 0.173 to 0.542], P = .001). Biomarkers of inflammation and oxidative stress are higher in individuals with asthma than without asthma, but the lower mtDNA-CN in asthma is independent of general inflammation or oxidative stress. Mendelian randomization studies suggest a potential causal relationship between asthma-associated genetic variants and mtDNA-CN., Conclusion: mtDNA-CN is lower in asthma than in no asthma and is associated with exacerbations. Low mtDNA-CN in asthma is not mediated through inflammation but is associated with a genetic predisposition to asthma., Competing Interests: Disclosure statement This research was conducted using the UK Biobank Resource under application 17731. The study was supported by the National Heart, Lung, and Blood Institute (NHLBI; HL081064, HL103453, and HL144569) and the National Center for Advancing Translational Sciences (ULTR000439). The Severe Asthma Research Program (SARP) was supported by awards from the National Heart, Lung, and Blood Institute (U10 HL109172, U10 HL109168, U10 HL109152, U10 HL109257, U10 HL109046, U10 HL109250, U10 HL109164, and U10 HL109086). SARP mitochondrial DNA copy numbers were from the Trans-omics in Precision Medicine (TOPMed) program supported by the NHLBI. Core support including centralized genomic read mapping and genotype calling, along with variant quality metrics and filtering, was provided by the TOPMed Informatics Research Center (3R01HL117626-02S1; contract HHSN268201800002I). Core support including phenotype harmonization, data management, sample-identity quality control, and general program coordination was provided by the TOPMed Data Coordinating Center (R01HL120393; U01HL120393; contract HHSN268201800001I). Industry support was provided for SARP III from AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi-Genzyme-Regeneron, and Teva. The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi-Genzyme-Regeneron, and Teva. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Many mouse models of SARS-CoV-2 infection involve expression of the human ACE2 protein, the entry receptor for SARS-CoV-2 Spike protein, in mouse tissues. However, most of these models suffer from nonphysiological regulation of ACE2 expression, which can lead to atypically severe infections and aberrant sites of viral replication. In this report, we developed and characterized an ACE2 gene replacement (ACE2-GR) mouse strain in which the mouse Ace2 genomic locus was replaced by the entire human ACE2 gene locus, and we investigated the ability of these animals to respond to SARS-CoV-2 infection. We show that ACE2-GR mice support SARS-CoV-2 viral replication, but, in stark contrast to the widely used K18-hACE2 transgenic model, this infection leads to a mild disease with no detectable involvement of the CNS. Thus, ACE2-GR mice provide a novel, to our knowledge, model to explore immune responses and long-term consequences of SARS-CoV-2 infection., (Copyright © 2024 The Authors.)

Background: The relative utility of eosinophil peroxidase (EPX) and blood and sputum eosinophil counts as disease biomarkers in asthma is uncertain., Objective: We sought to determine the utility of EPX as a biomarker of systemic and airway eosinophilic inflammation in asthma., Methods: EPX protein was measured by immunoassay in serum and sputum in 110 healthy controls to establish a normal reference range and in repeated samples of serum and sputum collected during 3 years of observation in 480 participants in the Severe Asthma Research Program 3., Results: Over 3 years, EPX levels in patients with asthma were higher than normal in 27% to 31% of serum samples and 36% to 53% of sputum samples. Eosinophils and EPX correlated better in blood than in sputum (r s values of 0.74 and 0.43, respectively), and high sputum EPX levels occurred in 27% of participants with blood eosinophil counts less than 150 cells/μL and 42% of participants with blood eosinophil counts between 150 and 299 cells/μL. Patients with persistently high sputum EPX values for 3 years were characterized by severe airflow obstruction, frequent exacerbations, and high mucus plug scores. In 59 patients with asthma who started mepolizumab during observation, serum EPX levels normalized in 96% but sputum EPX normalized in only 49%. Lung function remained abnormal even when sputum EPX normalized., Conclusions: Serum EPX is a valid protein biomarker of systemic eosinophilic inflammation in asthma, and sputum EPX levels are a more sensitive biomarker of airway eosinophilic inflammation than sputum eosinophil counts. Eosinophil measures in blood frequently miss airway eosinophilic inflammation, and mepolizumab frequently fails to normalize airway eosinophilic inflammation even though it invariably normalizes systemic eosinophilic inflammation., Competing Interests: Disclosure statement The study was supported by grants from the National Institutes of Health (grant nos. U10 HL109172, U10 HL109168, U10 HL109152, U10 HL109257, U10 HL109146, U10 HL109164, and U10 HL109086). The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi–Genzyme–Regeneron, and Teva. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative. Disclosure of potential conflict of interest: M. Tang has participated in advisory boards with Sanofi. M. W. Johansson has received grants from Hoffmann-La Roche. N. N. Jarjour has received consulting fees from GlaxoSmithKline. L. C. Denlinger has received grants from the American Lung Association-Airway Clinical Research Centers (ALA-ACRC); has received consulting fees from OM Pharma; and has received study drugs for the PrecISE trial from GlaxoSmithKline, Laurel, Sun Pharma, Vifor/OM Pharma Vitaeris/CSL Behring, and Vitaflo. M. C. Peters is an employee and has stock in Gilead Sciences. M. Castro has received grants from the ALA-ACRC, Patient-Centered Outcomes Research Institute (PCORI), AstraZeneca, Gala Therapeutics, Genentech, GlaxoSmithKline, Novartis, Pulmatrix, Sanofi-Aventis, Shinogi, and Theravance; has received consulting fees from Genentech, Teva, Sanofi-Aventis, Merck, Novartis, Arrowhead Pharmaceuticals, Allakos, Amgen, OM Pharma, Pfizer, Pioneering Medicines, and GlaxoSmithKline; has received honoraria for presentations from Amgen, AstraZeneca, Genentech, Regeneron, Sanofi-Aventis, and Teva; and has stock in Aer Therapeutics. K. Sumino has participated in advisory boards with AstraZeneca. B. D. Levy has received grants from Pleris Pharmaceuticals; has received consulting fees from AstraZeneca, Gossamer Bio, Novartis, and Thetis Pharmaceuticals; and has stock ownership in Entrinsic Health and Nocion Therapeutics. E. Israel has received grants from AstraZeneca, Avillion, Gossamer Bio, and PCORI; has received royalties from UpToDate—Wolters Kluwer; has received consulting fees from AB Science, Allergy and Asthma Network, Amgen, Arrowhead Pharmaceuticals, AstraZeneca, Avillion, GlaxoSmithKline, Merck, National Heart, Lung, and Blood Institute, Pneuma Respiratory, PPS Health, Regeneron, Sanofi-Genzyme, Teva, and Cowen; has received honoraria for presentations to Westchester Medical Center; has received payment for expert testimony to Cambridge Medical Experts, Danager Lagnese, and SettlePou; has participated in an advisory board with Novartis; is an unpaid member of the coordinating committee for the National Asthma Education and Prevention Program; has stock in Vorso; and has received equipment and study drugs from Circassia, Genentech, Teva, Sun Pharma, Laurel Pharmaceuticals, Om Pharma, Nestle, CSL Behring, and Sanofi-Regeneron. W. Phipatanakul has received grants from Sanofi, Regeneron, GlaxoSmithKline, Genentech, and Novartis; has received consulting fees from Sanofi, Regeneron, GlaxoSmithKline, Genentech, Novartis, and AstraZeneca; and has received support for attending meetings/travel from Sanofi and Regeneron. S. E. Wenzel has received grants from Regeneron; has received consulting fees from Sanofi and Novartis; and has stock in Aer Therapeutics. P. G. Woodruff has received consulting fees from Roche, Sanofi, Regeneron, and AstraZeneca. J. V. Fahy has received consulting fees and has stock from Suzhou Connect Biopharmaceuticals, Ltd, and Aer Therapeutics; and has been issued two patents. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that IL-13-activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibited viscoelastic liquid behavior and that mucus secreted by IL-13-activated HAECs exhibited solid-like behavior caused by mucin cross-linking. In addition, IL-13-activated HAECs shows increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that was prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), was increased in IL-13-activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings was increased in patients with asthma with high airway mucus plug scores. Together, our results show that IL-13-activated HAECs autonomously generated pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

Memory CD8 T cells (T mem ) can be activated into innate-like killers by cytokines like IL-12, IL-15, and/or IL-18; but mechanisms regulating this phenomenon (termed bystander activation) are not fully resolved. We found strain-intrinsic deficiencies in bystander activation using specific pathogen-free mice, whereby basal IL-4 signals antagonize IL-18 sensing. We show that therapeutic and helminth-induced IL-4 impairs protective bystander-mediated responses against pathogens. However, this IL-4/IL-18 axis does not completely abolish bystander activation but rather tunes the expression of direct versus indirect mediators of cytotoxicity (granzymes and interferon-γ, respectively). We show that antigen-experience overrides strain-specific deficiencies in bystander activation, leading to uniform IL-18 receptor expression and enhanced capacity for bystander activation/cytotoxicity. Our data highlight that bystander activation is not a binary process but tuned/deregulated by other cytokines that are elevated by contemporaneous infections. Further, our findings underscore the importance of antigen-experienced T mem to dissect the contributions of bystander T mem in health and disease.

Interleukin-7 (IL-7) is considered a critical regulator of memory CD8 + T cell homeostasis, but this is primarily based on analysis of circulating and not tissue-resident memory (T RM ) subsets. Furthermore, the cell-intrinsic requirement for IL-7 signaling during memory homeostasis has not been directly tested. Using inducible deletion, we found that Il7ra loss had only a modest effect on persistence of circulating memory and T RM subsets and that IL-7Rα was primarily required for normal basal proliferation. Loss of IL-15 signaling imposed heightened IL-7Rα dependence on memory CD8 + T cells, including T RM populations previously described as IL-15-independent. In the absence of IL-15 signaling, IL-7Rα was upregulated, and loss of IL-7Rα signaling reduced proliferation in response to IL-15, suggesting cross-regulation in memory CD8 + T cells. Thus, across subsets and tissues, IL-7 and IL-15 act in concert to support memory CD8 + T cells, conferring resilience to altered availability of either cytokine., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 ( n = 439) and from healthy controls ( n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation. NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Rationale: Density thresholds in computed tomography (CT) lung scans quantify air trapping (AT) at the whole-lung level but are not informative for AT in specific bronchopulmonary segments. Objectives: To apply a segment-based measure of AT in asthma to investigate the clinical determinants of AT in asthma. Methods: In each of 19 bronchopulmonary segments in CT lung scans from 199 patients with asthma, AT was categorized as present if lung attenuation was less than -856 Hounsfield units at expiration in ⩾15% of the lung area. The resulting AT segment score (0-19) was related to patient outcomes. Measurements and Main Results: AT varied at the lung segment level and tended to persist at the patient and lung segment levels over 3 years. Patients with widespread AT (⩾10 segments) had more severe asthma ( P  < 0.05). The mean (±SD) AT segment score in patients with a body mass index ⩾30 kg/m 2 was lower than in patients with a body mass index <30 kg/m 2 (3.5 ± 4.6 vs. 5.5 ± 6.3; P  = 0.008), and the frequency of AT in lower lobe segments in obese patients was less than in upper and middle lobe segments (35% vs. 46%; P  = 0.001). The AT segment score in patients with sputum eosinophils ⩾2% was higher than in patients without sputum eosinophilia (7.0 ± 6.1 vs. 3.3 ± 4.9; P  < 0.0001). Lung segments with AT more frequently had airway mucus plugging than lung segments without AT (48% vs. 18%; P  ⩽ 0.0001). Conclusions: In patients with asthma, air trapping is more severe in those with airway eosinophilia and mucus plugging, whereas those who are obese have less severe trapping because their lower lobe segments are spared.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC + mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma., (© 2024. The Author(s).)

Airway inflammation in asthma has been well recognized for several decades, with general agreement on its role in asthma pathogenesis, symptoms, propensity toward exacerbation, and decline in lung function. This has led to universal recommendation in asthma management guidelines to incorporate the use of inhaled corticosteroid as an anti-inflammatory therapy for all patients with persistent asthma symptoms. However, there has been limited attention paid to the presence and potential impact of systemic inflammation in asthma. Accumulating evidence from epidemiological observations and cohort studies points to a host of downstream organ dysfunction in asthma especially among patients with longstanding or more severe disease, frequent exacerbations, and underlying risk factors for organ dysfunction. Most studies to date have focused on cognitive impairment, depression/anxiety, metabolic syndrome, and cardiovascular abnormalities. In this review, we summarize some of the evidence demonstrating these abnormalities and highlight the proposed mechanisms and potential benefits of treatment in limiting these extrapulmonary abnormalities in patients with asthma. The goal of this commentary is to raise awareness of the importance of recognizing potential extrapulmonary conditions associated with systemic inflammation of asthma. This area of treatment of patients with asthma is a large unmet need., (Copyright © 2024. Published by Elsevier Inc.)

BACKGROUNDInformation about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments.METHODSCT lung scans from 57 patients with asthma were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow.RESULTSThe length distribution of 778 annotated mucus plugs was multimodal, and a 12 mm length defined short ("stubby", ≤12 mm) and long ("stringy", >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6-9, and 47% of plugs in baseline scans persisted in the same airway for 3 years and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted a greater effect for proximal generations and more numerous mucus plugs.CONCLUSIONPersistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy.TRIAL REGISTRATIONClinicaltrials.gov; NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01716494, and NCT01760915.FUNDINGAstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi-Genzyme-Regeneron, and TEVA provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.

Background: Accumulating evidence indicates that asthma has systemic effects and affects brain function. Although airway inflammation is proposed to initiate afferent communications with the brain, the signaling pathways have not been established., Objective: We sought to identify the cellular and molecular pathways involved in afferent lung-brain communication during airway inflammation in asthma., Methods: In 23 adults with mild asthma, segmental bronchial provocation with allergen (SBP-Ag) was used to provoke airway inflammation and retrieve bronchoalveolar lavage fluid for targeted protein analysis and RNA sequencing to determine gene expression profiles. Neural responses to emotional cues in nodes of the salience network were assessed with functional magnetic resonance imaging at baseline and 48 hours after SBP-Ag., Results: Cell deconvolution and gene coexpression network analysis identified 11 cell-associated gene modules that changed in response to SBP-Ag. SBP-Ag increased bronchoalveolar lavage eosinophils and expression of an eosinophil-associated module enriched for genes related to T H 17-type inflammation (eg, IL17A), as well as cell proliferation in lung and brain (eg, NOTCH1, VEGFA, and LIF). Increased expression of genes in this module, as well as several T H 17-type inflammation-related proteins, was associated with an increase from baseline in salience network reactivity., Conclusions: Our results identify a specific inflammatory pathway linking asthma-related airway inflammation and emotion-related neural function. Systemically, T H 17-type inflammation has been implicated in both depression and neuroinflammation, with impacts on long-term brain health. Thus, our data emphasize that inflammation in the lung in asthma may have profound effects outside of the lung that may be targetable with novel therapeutic approaches., Competing Interests: Disclosure statement This work was supported by the National Heart, Lung, and Blood Institute (grant no. R01HL123284 to W.W.B.). Disclosure of potential conflict of interest: M. C. Altman reports personal fees from Sanofi-Regeneron, outside the submitted work. W. W. Busse reports consulting fees/honoraria from GlaxoSmithKline, Novartis, AstraZeneca, Regeneron, Sanofi, and Genentech; and royalties from Elsevier. N. N. Jarjour received research funding from AstraZeneca for extension of the National Institutes of Health–funded severe asthma research program; and received consulting fees over the past 3 years (<$5000) from GlaxoSmithKline with regard to severe asthma treatment. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The recall periods and response scales of existing surveys of asthma control are poorly suited for studying acute exacerbations., Objective: To develop an instrument able to predict exacerbations after the onset of acute symptoms and with a recall window sufficiently short to study recovery., Methods: We developed the six-item Acute Asthma Exacerbation Survey (AAES). Data were collected at baseline, acute, and recovery visits within an established longitudinal protocol for participants with severe asthma. Participants scheduled acute study visits at the first sign of a cold. Nasal lavage samples and lung function measurements were also collected. The AAES data were analyzed using Cronbach α, Spearman correlations, and Kruskal-Wallace methods. We used logistic regression for predictors of bursts of oral corticosteroids (OCS)., Results: Of 130 participants studied at baseline, 52 returned for an acute visit. The AAES scores were elevated at the acute visit and returned to baseline after recovery independently of respiratory virus detection. Cronbach α for the AAES was 0.853, 0.822, and 0.889 at the three respective visits. Compared with participants not needing burst OCS, those with exacerbations had higher acute AAES scores (16 [13.5-18] vs 11.5 [8.2-14], median [interquartile range]; P = .017) and a larger reduction from baseline in lung function. For each 3-point increase in AAES scores, the odds ratio for burst OCS use was 1.64 (95% CI, 1.04-2.57; P = .030)., Conclusions: The AAES is internally consistent and dynamically responsive during acute asthma exacerbations. Additional validation studies are warranted to support future trials and aid in clinical decision-making., (Copyright © 2023. Published by Elsevier Inc.)

TH17 cells are implicated in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). We previously reported that the transcription factor basic helix-loop-helix family member e40 (BHLHE40) marks cytokine-producing pathogenic TH cells during EAE, and that its expression in T cells is required for clinical disease. In this study, using dual reporter mice, we show BHLHE40 expression within TH1/17 and ex-TH17 cells following EAE induction. Il17a-Cre-mediated deletion of BHLHE40 in TH cells led to less severe EAE with reduced TH cell cytokine production. Characterization of the leukocytes in the CNS during EAE by single-cell RNA sequencing identified differences in the infiltrating myeloid cells when BHLHE40 was present or absent in TH17 cells. Our studies highlight the importance of BHLHE40 in promoting TH17 cell encephalitogenicity and instructing myeloid cell responses during active EAE., (Copyright © 2023 The Authors.)

Objective: Genome-wide association studies (GWASs) have identified single nucleotide polymorphisms (SNPs) in chr11p15.5 region associated with asthma and idiopathic interstitial pneumonias (IIPs). We sought to identify functional genes for asthma by combining SNPs and mRNA expression in bronchial epithelial cells (BEC) in the Severe Asthma Research Program (SARP)., Methods: Correlation analyses of mRNA expression of six candidate genes ( AP2A2 , MUC6 , MUC2 , MUC5AC , MUC5B , and TOLLIP ) and asthma phenotypes were performed in the longitudinal cohort ( n  = 156) with RNAseq in BEC, and replicated in the cross-sectional cohort ( n  = 155). eQTL ( n  = 114) and genetic association analysis of asthma severity (426 severe vs. 531 non-severe asthma) were performed, and compared with previously published GWASs of IIPs and asthma., Results: Higher expression of AP2A2 and MUC5AC and lower expression of MUC5B in BEC were correlated with asthma, asthma exacerbations, and T2 biomarkers ( P  < 0.01). SNPs associated with asthma and IIPs in previous GWASs were eQTL SNPs for MUC5AC , MUC5B , or TOLLIP , however, they were not in strong linkage disequilibrium. The risk alleles for asthma or protective alleles for IIPs were associated with higher expression of MUC5AC and lower expression of MUC5B . rs11603634, rs12788104, and rs28415845 associated with moderate-to-severe asthma or adult onset asthma in previous GWASs were not associated with asthma severity ( P  > 0.8)., Conclusions: SNPs associated with asthma in chr11p15.5 region are not associated with asthma severity neither with IIPs. Higher expression of MUC5AC and lower expression of MUC5B are risk for asthma but protective for IIPs.

Objective: Subphenotypes of asthma may be determined by age onset and atopic status. We sought to characterize early or late onset atopic asthma with fungal or non-fungal sensitization (AAFS or AANFS) and non-atopic asthma (NAA) in children and adults in the Severe Asthma Research Program (SARP). SARP is an ongoing project involving well-phenotyped patients with mild to severe asthma., Methods: Phenotypic comparisons were performed using Kruskal-Wallis or chi-square test. Genetic association analyses were performed using logistic or linear regression., Results: Airway hyper-responsiveness, total serum IgE levels, and T2 biomarkers showed an increasing trend from NAA to AANFS and then to AAFS. Children and adults with early onset asthma had greater % of AAFS than adults with late onset asthma (46% and 40% vs. 32%; P  < 0.00001). In children, AAFS and AANFS had lower % predicted FEV 1 (86% and 91% vs. 97%) and greater % of patients with severe asthma than NAA (61% and 59% vs. 43%). In adults with early or late onset asthma, NAA had greater % of patients with severe asthma than AANFS and AAFS (61% vs. 40% and 37% or 56% vs. 44% and 49%). The G allele of rs2872507 in GSDMB had higher frequency in AAFS than AANFS and NAA (0.63 vs. 0.55 and 0.55), and associated with earlier age onset and asthma severity., Conclusions: Early or late onset AAFS, AANFS, and NAA have shared and distinct phenotypic characteristics in children and adults. AAFS is a complex disorder involving genetic susceptibility and environmental factors.

Purpose: The purpose of this study was to anatomically correlate ventilation defects with regions of air trapping by whole lung, lung lobe, and airway segment in the context of airway mucus plugging in asthma. Methods: A total of 34 asthmatics [13M:21F, 13 mild/moderate, median age (range) of 49.5 (36.8-53.3) years and 21 severe, 56.1 (47.1-62.6) years] and 4 healthy subjects [1M:3F, 38.5 (26.6-52.2) years] underwent HP 3 He MRI and CT imaging. HP 3 He MRI was assessed for ventilation defects using a semi-automated k-means clustering algorithm. Inspiratory and expiratory CTs were analyzed using parametric response mapping (PRM) to quantify markers of emphysema and functional small airways disease (fSAD). Segmental and lobar lung masks were obtained from CT and registered to HP 3 He MRI in order to localize ventilation defect percent (VDP), at the lobar and segmental level, to regions of fSAD and mucus plugging. Spearman's correlation was utilized to compare biomarkers on a global and lobar level, and a multivariate analysis was conducted to predict segmental fSAD given segmental VDP (sVDP) and mucus score as variables in order to further understand the functional relationships between regional measures of obstruction. Results: On a global level, fSAD was correlated with whole lung VDP ( r = 0.65, p < 0.001), mucus score ( r = 0.55, p < 0.01), and moderately correlated (-0.60 ≤ r ≤ -0.56, p < 0.001) to percent predicted (%p) FEV1, FEF25-75 and FEV1/FVC, and more weakly correlated to FVC%p (-0.38 ≤ r ≤ -0.35, p < 0.001) as expected from previous work. On a regional level, lobar VDP, mucus scores, and fSAD were also moderately correlated (r from 0.45-0.66, p < 0.01). For segmental colocalization, the model of best fit was a piecewise quadratic model, which suggests that sVDP may be increasing due to local airway obstruction that does not manifest as fSAD until more extensive disease is present. sVDP was more sensitive to the presence of a mucus plugs overall, but the prediction of fSAD using multivariate regression showed an interaction in the presence of a mucus plugs when sVDP was between 4% and 10% ( p < 0.001). Conclusion: This multi-modality study in asthma confirmed that areas of ventilation defects are spatially correlated with air trapping at the level of the airway segment and suggests VDP and fSAD are sensitive to specific sources of airway obstruction in asthma, including mucus plugs., Competing Interests: SF receives research support from GE Healthcare and serves on the scientific advisory board of Polarean PLC. KC and CH are currently employees of Imbio LLC. LD and NJ are Principal Investigators in the NHLBI PrecISE Trial network that has relationships with the following companies for drug support and equipment contracts: Glaxo Smith Kline, Laurel, Sun Pharma, Vifor/OM Pharm, Vitaeris/CSL Behring, Vitaflo, Vyaire, Caire Diagnostics, MIR, Propeller Health, ZEPHYRx. NJ reports grants and personal fees from AstraZeneca, and personal fees from GSK and Boehringer Ingelheim for consultations outside the submitted work. LD has consulted with Glaxo Smith Kline, AstraZeneca, Sanofi and OM Pharma during the course of this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Carey, Hotvedt, Mummy, Lee, Denlinger, Schiebler, Sorkness, Jarjour, Hatt, Galban and Fain.)

The 5' untranslated regions (UTRs) in messenger RNAs (mRNAs) play an important role in the regulation of protein synthesis. We had previously identified a group of mRNAs that includes human semaphorin 7A (SEMA7A) whose translation is upregulated by the Erk/p90S6K pathway in human eosinophils, with a potential negative impact in asthma and airway inflammation. In the current study, we aimed to find a common 5'UTR regulatory cis-element, and determine its impact on protein synthesis. We identified a common and conserved 5'UTR motif GGCTG-[(C/G)T(C/G)] n -GCC that was present in this group of mRNAs. Mutations of the first two GG bases in this motif in SEMA7A 5'UTR led to a complete loss of S6K activity dependence for maximal translation. In conclusion, the newly identified 5'UTR motif present in SEMA7A has a critical role in regulating S6K-dependent protein synthesis., Competing Interests: Declaration of competing interest Nizar N. Jarjour has received consulting fees from Glaxo Smith Kline (GSK), Astra-Zeneca, and Boehringer Ingelheim over the past three years. These relationships with pharmaceutical companies are not relevant to the current study. The remaining authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Inhaled corticosteroids (CSs) are the backbone of asthma treatment, improving quality of life, exacerbation rates, and mortality. Although effective for most, a subset of patients with asthma experience CS-resistant disease despite receiving high-dose medication., Objective: We sought to investigate the transcriptomic response of bronchial epithelial cells (BECs) to inhaled CSs., Methods: Independent component analysis was performed on datasets, detailing the transcriptional response of BECs to CS treatment. The expression of these CS-response components was examined in 2 patient cohorts and investigated in relation to clinical parameters. Supervised learning was used to predict BEC CS responses using peripheral blood gene expression., Results: We identified a signature of CS response that was closely correlated with CS use in patients with asthma. Participants could be separated on the basis of CS-response genes into groups with high and low signature expression. Patients with low expression of CS-response genes, particularly those with a severe asthma diagnosis, showed worse lung function and quality of life. These individuals demonstrated enrichment for T-lymphocyte infiltration in endobronchial brushings. Supervised machine learning identified a 7-gene signature from peripheral blood that reliably identified patients with poor CS-response expression in BECs., Conclusions: Loss of CS transcriptional responses within bronchial epithelium was related to impaired lung function and poor quality of life, particularly in patients with severe asthma. These individuals were identified using minimally invasive blood sampling, suggesting these findings may enable earlier triage to alternative treatments., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Rationale: CC16 is a protein mainly produced by nonciliated bronchial epithelial cells (BECs) that participates in host defense. Reduced CC16 protein concentrations in BAL and serum are associated with asthma susceptibility. Objectives: Few studies have investigated the relationship between CC16 and asthma progression, and none has focused on BECs. In this study, we sought to determine if CC16 mRNA expression levels in BECs are associated with asthma severity. Methods: Association analyses between CC16 mRNA expression levels in BECs (242 asthmatics and 69 control subjects) and asthma-related phenotypes in Severe Asthma Research Program were performed using a generalized linear model. Measurements and Main Results: Low CC16 mRNA expression levels in BECs were significantly associated with asthma susceptibility and asthma severity, high systemic corticosteroids use, high retrospective and prospective asthma exacerbations, and low pulmonary function. Low CC16 mRNA expression levels were significantly associated with high T2 inflammation biomarkers (fractional exhaled nitric oxide and sputum eosinophils). CC16 mRNA expression levels were negatively correlated with expression levels of Th2 genes ( IL1RL1 , POSTN , SERPINB2 , CLCA1 , NOS2 , and MUC5AC ) and positively correlated with expression levels of Th1 and inflammation genes ( IL12A and MUC5B ). A combination of two nontraditional T2 biomarkers (CC16 and IL-6) revealed four asthma endotypes with different characteristics of T2 inflammation, obesity, and asthma severity. Conclusions: Our findings indicate that low CC16 mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations, partially through immunomodulation of T2 inflammation. CC16 is a potential nontraditional T2 biomarker for asthma development and progression.

Rationale: Extrapulmonary manifestations of asthma, including fatty infiltration in tissues, may reflect systemic inflammation and influence lung function and disease severity. Objectives: To determine if skeletal muscle adiposity predicts lung function trajectory in asthma. Methods: Adult SARP III (Severe Asthma Research Program III) participants with baseline computed tomography imaging and longitudinal postbronchodilator FEV 1 % predicted (median follow-up 5 years [1,132 person-years]) were evaluated. The mean of left and right paraspinous muscle density (PSMD) at the 12th thoracic vertebral body was calculated (Hounsfield units [HU]). Lower PSMD reflects higher muscle adiposity. We derived PSMD reference ranges from healthy control subjects without asthma. A linear multivariable mixed-effects model was constructed to evaluate associations of baseline PSMD and lung function trajectory stratified by sex. Measurements and Main Results: Participants included 219 with asthma (67% women; mean [SD] body mass index, 32.3 [8.8] kg/m 2 ) and 37 control subjects (51% women; mean [SD] body mass index, 26.3 [4.7] kg/m 2 ). Participants with asthma had lower adjusted PSMD than control subjects (42.2 vs. 55.8 HU; P  < 0.001). In adjusted models, PSMD predicted lung function trajectory in women with asthma (β = -0.47 Δ slope per 10-HU decrease; P  = 0.03) but not men (β = 0.11 Δ slope per 10-HU decrease; P  = 0.77). The highest PSMD tertile predicted a 2.9% improvement whereas the lowest tertile predicted a 1.8% decline in FEV 1 % predicted among women with asthma over 5 years. Conclusions: Participants with asthma have lower PSMD, reflecting greater muscle fat infiltration. Baseline PSMD predicted lung function decline among women with asthma but not men. These data support an important role of metabolic dysfunction in lung function decline.

Asthma is characterized by airflow limitations resulting from bronchial closure, which can be either reversible or fixed due to changes in airway tissue composition and structure, also known as remodeling. Airway remodeling is defined as increased presence of mucins-producing epithelial cells, increased thickness of airway smooth muscle cells, angiogenesis, increased number and activation state of fibroblasts, and extracellular matrix (ECM) deposition. Airway inflammation is believed to be the main cause of the development of airway remodeling in asthma. In this chapter, we will review the development of the adaptive immune response and the impact of its mediators and cells on the elements defining airway remodeling in asthma., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background Asthma and atherosclerotic cardiovascular disease share an underlying inflammatory pathophysiology. We hypothesized that persistent asthma is associated with carotid plaque burden, a strong predictor of atherosclerotic cardiovascular disease events. Methods and Results The MESA (Multi-Ethnic Study of Atherosclerosis) enrolled adults free of known atherosclerotic cardiovascular disease at baseline. Subtype of asthma was determined at examination 1. Persistent asthma was defined as asthma requiring use of controller medications, and intermittent asthma was defined as asthma without controller medications. B-mode carotid ultrasound was performed to detect carotid plaques (total plaque score [TPS], range 0-12). Multivariable regression modeling with robust variances evaluated the association of asthma subtype and carotid plaque burden. The 5029 participants were a mean (SD) age of 61.6 (10.0) years (53% were women, 26% were Black individuals, 23% were Hispanic individuals, and 12% were Chinese individuals). Carotid plaque was present in 50.5% of participants without asthma (TPS, 1.29 [1.80]), 49.5% of participants with intermittent asthma (TPS, 1.25 [1.76]), and 67% of participants with persistent asthma (TPS, 2.08 [2.35]) ( P ≤0.003). Participants with persistent asthma had higher interleukin-6 (1.89 [1.61] pg/mL) than participants without asthma (1.52 [1.21] pg/mL; P =0.02). In fully adjusted models, persistent asthma was associated with carotid plaque presence (odds ratio, 1.83 [95% confidence interval, 1.21-2.76]; P <0.001) and TPS (β=0.66; P <0.01), without attenuation after adjustment for baseline interleukin-6 ( P =0.02) or CRP (C-reactive protein) ( P =0.01). Conclusions Participants with persistent asthma had higher carotid plaque burden and higher levels of inflammatory biomarkers, compared with participants without asthma. Adjustment for baseline inflammatory biomarkers did not attenuate the association between carotid plaque and asthma subtype, highlighting the increased atherosclerotic cardiovascular disease risk among those with persistent asthma may be multifactorial.

Asthma is an inflammatory disease of the airways characterized by eosinophil recruitment, eosinophil peroxidase release, and protein oxidation through bromination, which following tissue remodeling results in excretion of 3-bromotyrosine. Predicting exacerbations and reducing their frequency is critical for the treatment of severe asthma. In this study, we aimed to investigate whether urinary total conjugated bromotyrosine can discriminate asthma severity and predict asthma exacerbations. We collected urine from participants with severe ( n = 253) and nonsevere ( n = 178) asthma, and the number of adjudicated exacerbations in 1-yr longitudinal follow-up was determined among subjects enrolled in the Severe Asthma Research Program, a large-scale National Institutes of Health (NIH)-funded consortium. Urine glucuronidated bromotyrosine and total conjugated forms were quantified by hydrolysis with either glucuronidase or methanesulfonic acid, respectively, followed by liquid chromatography-tandem mass spectrometry analyses of free 3-bromotyrosine. Blood and sputum eosinophils were also counted. The majority of 3-bromotyrosine in urine was found to exist in conjugated forms, with glucuronidated bromotyrosine representing approximately a third, and free bromotyrosine less than 1% of total conjugated bromotyrosine. Total conjugated bromotyrosine was poorly correlated with blood ( r 2 = 0.038) or sputum eosinophils ( r 2 = 0.0069). Compared with participants with nonsevere asthma, participants with severe asthma had significantly higher urinary total conjugated bromotyrosine levels. Urinary total conjugated bromotyrosine was independently associated with asthma severity, correlated with the number of asthma exacerbations, and served as a predictor of asthma exacerbation risk over 1-yr of follow-up.

Rationale: The role of obesity-associated insulin resistance (IR) in airflow limitation in asthma is uncertain. Objectives: Using data in the Severe Asthma Research Program 3 (SARP-3), we evaluated relationships between homeostatic measure of IR (HOMA-IR), lung function (cross-sectional and longitudinal analyses), and treatment responses to bronchodilators and corticosteroids. Methods: HOMA-IR values were categorized as without (<3.0), moderate (3.0-5.0), or severe (>5.0). Lung function included FEV 1 and FVC measured before and after treatment with inhaled albuterol and intramuscular triamcinolone acetonide and yearly for 5 years. Measurements and Main Results: Among 307 participants in SARP-3, 170 (55%) were obese and 140 (46%) had IR. Compared with patients without IR, those with IR had significantly lower values for FEV 1 and FVC, and these lower values were not attributable to obesity effects. Compared with patients without IR, those with IR had lower FEV 1 responses to β-adrenergic agonists and systemic corticosteroids. The annualized decline in FEV 1 was significantly greater in patients with moderate IR (-41 ml/year) and severe IR (-32 ml/year,) than in patients without IR (-13 ml/year, P  < 0.001 for both comparisons). Conclusions: IR is common in asthma and is associated with lower lung function, accelerated loss of lung function, and suboptimal lung function responses to bronchodilator and corticosteroid treatments. Clinical trials in patients with asthma and IR are needed to determine if improving IR might also improve lung function.

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8 + T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15-independent CD8 + T cell memory populations, including tissue-resident memory CD8 + T cells (T RM ) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15-insensitive memory CD8 + T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8 + T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rβ. This was conserved for memory CD8 + T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c-induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplify memory populations. Through parabiosis, we showed that IL-15c drive local proliferation of T RM , with a degree of recruitment of circulating cells to some NLTs. Hence, irrespective of homeostatic IL-15 dependence, IL-15 sensitivity is a defining feature of memory CD8 + T cell populations, with therapeutic potential for expansion of T RM and other memory subsets in an antigen-agnostic and temporally controlled fashion.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling., Competing Interests: The authors acknowledge the following potential conflicts of interest in companies pursuing open microfluidic and biomedical technologies: EB: Tasso, Inc., Salus Discovery, LLC, and Stacks to the Future, LLC; AT: Stacks to the Future, LLC. However, the research in this publication is not related to these companies. SM is a consultant and speaker for Astra-Zeneca and GlaxoSmithKline. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zeng, Su, Takezawa, Fichtinger, Lee, Pippin, Shankland, Lim, Denlinger, Jarjour, Mathur, Sandbo, Berthier, Esnault, Bernau and Theberge.)

The local microenvironment shapes macrophage differentiation in each tissue. We hypothesized that in the peritoneum, local factors in addition to retinoic acid can support GATA6-driven differentiation and function of peritoneal large cavity macrophages (LCMs). We found that soluble proteins produced by mesothelial cells lining the peritoneal cavity maintained GATA6 expression in cultured LCMs. Analysis of global gene expression of isolated mesothelial cells highlighted mesothelin (Msln) and its binding partner mucin 16 (Muc16) as candidate secreted ligands that potentially regulate GATA6 expression in peritoneal LCMs. Mice deficient for either of these molecules showed diminished GATA6 expression in peritoneal and pleural LCMs that was most prominent in aged mice. The more robust phenotype in older mice suggested that monocyte-derived macrophages were the target of Msln and Muc16. Cell transfer and bone marrow chimera experiments supported this hypothesis. We found that lethally irradiated Msln-/- and Muc16-/- mice reconstituted with wild-type bone marrow had lower levels of GATA6 expression in peritoneal and pleural LCMs. Similarly, during the resolution of zymosan-induced inflammation, repopulated peritoneal LCMs lacking expression of Msln or Muc16 expressed diminished GATA6. These data support a role for mesothelial cell-produced Msln and Muc16 in local macrophage differentiation within large cavity spaces such as the peritoneum. The effect appears to be most prominent on monocyte-derived macrophages that enter into this location as the host ages and also in response to infection., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background Clustering key clinical characteristics of participants in the Severe Asthma Research Program (SARP), a large, multicenter prospective observational study of patients with asthma and healthy controls, has led to the identification of novel asthma phenotypes. Purpose To determine whether quantitative CT (qCT) could help distinguish between clinical asthma phenotypes. Materials and Methods A retrospective cross-sectional analysis was conducted with the use of qCT images (maximal bronchodilation at total lung capacity [TLC], or inspiration, and functional residual capacity [FRC], or expiration) from the cluster phenotypes of SARP participants (cluster 1: minimal disease; cluster 2: mild, reversible; cluster 3: obese asthma; cluster 4: severe, reversible; cluster 5: severe, irreversible) enrolled between September 2001 and December 2015. Airway morphometry was performed along standard paths (RB1, RB4, RB10, LB1, and LB10). Corresponding voxels from TLC and FRC images were mapped with use of deformable image registration to characterize disease probability maps (DPMs) of functional small airway disease (fSAD), voxel-level volume changes (Jacobian), and isotropy (anisotropic deformation index [ADI]). The association between cluster assignment and qCT measures was evaluated using linear mixed models. Results A total of 455 participants were evaluated with cluster assignments and CT (mean age ± SD, 42.1 years ± 14.7; 270 women). Airway morphometry had limited ability to help discern between clusters. DPM fSAD was highest in cluster 5 (cluster 1 in SARP III: 19.0% ± 20.6; cluster 2: 18.9% ± 13.3; cluster 3: 24.9% ± 13.1; cluster 4: 24.1% ± 8.4; cluster 5: 38.8% ± 14.4; P < .001). Lower whole-lung Jacobian and ADI values were associated with greater cluster severity. Compared to cluster 1, cluster 5 lung expansion was 31% smaller (Jacobian in SARP III cohort: 2.31 ± 0.6 vs 1.61 ± 0.3, respectively, P < .001) and 34% more isotropic (ADI in SARP III cohort: 0.40 ± 0.1 vs 0.61 ± 0.2, P < .001). Within-lung Jacobian and ADI SDs decreased as severity worsened (Jacobian SD in SARP III cohort: 0.90 ± 0.4 for cluster 1; 0.79 ± 0.3 for cluster 2; 0.62 ± 0.2 for cluster 3; 0.63 ± 0.2 for cluster 4; and 0.41 ± 0.2 for cluster 5; P < .001). Conclusion Quantitative CT assessments of the degree and intraindividual regional variability of lung expansion distinguished between well-established clinical phenotypes among participants with asthma from the Severe Asthma Research Program study. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Verschakelen in this issue.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

Background: Airway remodeling in patients with asthma, which leads to a decline in pulmonary function, is likely the result of repeated exacerbations often provoked by aeroallergen exposures. Aeroallegen exposure triggers a stereotypic response orchestrated by growth factor cytokines and other protein mediators. This results in a late-phase allergic reaction characterized by vascular permeability, recruitment of activated leukocytes, and activation of structural cells of the airway. The spectrum of protein mediators and their functions are incompletely understood., Methods: Bronchoalveolar lavage fluid (BALF) samples were obtained from 12 volunteers who exhibited robust eosinophilic recruitment following segmental bronchial provocation with allergen (SBP-Ag). We systematically identified and quantified proteins in BALF using high-performance liquid chromatography-high-resolution mass spectrometry (LC-MS/MS) followed by pathway analysis and correlations with airway physiology., Results: Pairwise analysis of protein abundance in BALF pre- vs post-SBP-Ag revealed that 55 proteins were upregulated and 103 proteins were downregulated. We observed enrichment of groups of proteins mapping to hemostasis/fibrin clot, platelet activation, lipoprotein assembly, neutrophil degranulation proteins, and acute-phase inflammation-airway remodeling pathways. The abundances of F2 and Fibrinogen γ (FGG) correlated with eosinophil numbers, whereas SERPINA3 negatively correlated with change in FeNO. The coagulation proteins F2 and KNG negatively correlated with FN1 an index of airway remodeling. Interestingly, patients with lower FEV 1 showed distinct allergen-induced patterns of 8 BALF proteins, including MUC1, alarmins (HSPB1), and actin polymerization factors., Conclusions: Protein abundance of the fibrin formation cascade, platelet activation and remodeling are associated with late-phase leukocyte numbers and markers of remodeling. Patients with lower FEV 1 have distinct dynamic responses to allergen., (© 2022. The Author(s).)

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell-dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti-PD-1 and anti-CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen-specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti-PD-1 and anti-CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome-tumor outgrowth-several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target., (©2022 The Authors; Published by the American Association for Cancer Research.)

Rationale: Cross-sectional analysis of mucus plugs in computed tomography (CT) lung scans in the Severe Asthma Research Program (SARP)-3 showed a high mucus plug phenotype. Objectives: To determine if mucus plugs are a persistent asthma phenotype and if changes in mucus plugs over time associate with changes in lung function. Methods: In a longitudinal analysis of baseline and Year 3 CT lung scans in SARP-3 participants, radiologists generated mucus plug scores to assess mucus plug persistence over time. Changes in mucus plug score were analyzed in relation to changes in lung function and CT air trapping measures. Measurements and Main Results: In 164 participants, the mean (range) mucus plug score was similar at baseline and Year 3 (3.4 [0-20] vs. 3.8 [0-20]). Participants and bronchopulmonary segments with a baseline plug were more likely to have plugs at Year 3 than those without baseline plugs (risk ratio, 2.8; 95% confidence interval [CI], 2.0-4.1; P  < 0.001; and risk ratio, 5.0; 95% CI, 4.5-5.6; P  < 0.001, respectively). The change in mucus plug score from baseline to Year 3 was significantly negatively correlated with change in FEV 1 % predicted ( r p  = -0.35; P  < 0.001) and with changes in CT air trapping measures (all P values < 0.05). Conclusions: Mucus plugs identify a persistent asthma phenotype, and susceptibility to mucus plugs occurs at the subject and the bronchopulmonary segment level. The association between change in mucus plug score and change in airflow over time supports a causal role for mucus plugs in mechanisms of airflow obstruction in asthma.

Asthma is a complex syndrome associated with episodic decompensations provoked by aeroallergen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bronchoalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial allergen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by pathway analysis and correlation with airway inflammation. SBP-Ag induced statistically significant changes in 549 features that mapped to 72 uniquely identified metabolites. From these features, two distinct inducible metabolic phenotypes were identified by the principal component analysis, partitioning around medoids (PAM) and k-means clustering. Ten index metabolites were identified that informed the presence of asthma-relevant pathways, including unsaturated fatty acid production/metabolism, mitochondrial beta oxidation of unsaturated fatty acid, and bile acid metabolism. Pathways were validated using proteomics in eosinophils. A segmental bronchial allergen challenge induces distinct metabolic responses in humans, providing insight into pathogenic and protective endotypes in allergic asthma.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases., Competing Interests: Declaration of interests The authors disclose no conflicts., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background Airway mucus plugs in asthma are associated with exacerbation frequency, increased eosinophilia, and reduced lung function. The relationship between mucus plugs and spatially overlapping ventilation abnormalities observed at hyperpolarized gas MRI has not been assessed quantitatively. Purpose To assess regional associations between CT mucus plugs scored by individual bronchopulmonary segment and corresponding measurements of segmental ventilation defect percentage (VDP) at hyperpolarized helium 3 ( 3 He) MRI. Materials and Methods In this secondary analysis of a Health Insurance Portability and Accountability Act-compliant prospective observational cohort, participants in the Severe Asthma Research Program (SARP) III (NCT01760915) between December 2012 and August 2015 underwent hyperpolarized 3 He MRI to determine segmental VDP. Segmental mucus plugs at CT were scored by two readers, with segments scored as plugged only if both readers agreed independently. A linear mixed-effects model controlling for interpatient variability was then used to assess differences in VDP in plugged versus plug-free segments. Results Forty-four participants with asthma were assessed (mean age ± standard deviation, 47 years ± 15; 29 women): 19 with mild-to-moderate asthma and 25 with severe asthma. Mucus plugs were observed in 49 total bronchopulmonary segments across eight of 44 patients. Segments containing mucus plugs had a median segmental VDP of 25.9% (25th-75th percentile, 7.3%-38.3%) versus 1.4% (25th-75th percentile, 0.1%-5.2%; P < .001) in plug-free segments. Similarly, the model estimated a segmental VDP of 18.9% (95% CI: 15.7, 22.2) for mucus-plugged segments versus 5.1% (95% CI: 3.3, 7.0) for plug-free segments ( P < .001). Participants with one or more mucus plugs had a median whole-lung VDP of 11.1% (25th-75th percentile, 7.1%-18.9%) versus 3.1% (25th-75th percentile, 1.1%-4.4%) in those without plugs ( P < .001). Conclusion Airway mucus plugging at CT was associated with reduced ventilation in the same bronchopulmonary segment at hyperpolarized helium 3 MRI, suggesting that mucus plugging may be an important cause of ventilation defects in asthma. © RSNA, 2021 Online supplemental material is available for this article.

Asthma is a heterogeneous disease, with multiple underlying inflammatory pathways and structural airway abnormalities that impact disease persistence and severity. Recent progress has been made in developing targeted asthma therapeutics, especially for subjects with eosinophilic asthma. However, there is an unmet need for new approaches to treat patients with severe and exacerbation-prone asthma, who contribute disproportionately to disease burden. Extensive deep phenotyping has revealed the heterogeneous nature of severe asthma and identified distinct disease subtypes. A current challenge in the field is to translate new and emerging knowledge about different pathobiologic mechanisms in asthma into patient-specific therapies, with the ultimate goal of modifying the natural history of disease. Here, we describe the Precision Interventions for Severe and/or Exacerbation-Prone Asthma (PrecISE) Network, a groundbreaking collaborative effort of asthma researchers and biostatisticians from around the United States. The PrecISE Network was designed to conduct phase II/proof-of-concept clinical trials of precision interventions in the population with severe asthma, and is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health. Using an innovative adaptive platform trial design, the PrecISE Network will evaluate up to 6 interventions simultaneously in biomarker-defined subgroups of subjects. We review the development and organizational structure of the PrecISE Network, and choice of interventions being studied. We hope that the PrecISE Network will enhance our understanding of asthma subtypes and accelerate the development of therapeutics for severe asthma., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Epidemiologic studies have shown that Alzheimer's disease (AD) and related dementias (ADRD) are seen more frequently with asthma, especially with greater asthma severity or exacerbation frequency., Objective: To examine the changes in brain structure that may underlie this phenomenon, we examined diffusion-weighted magnetic resonance imaging (dMRI) and blood-based biomarkers of AD (phosphorylated tau 181, p-Tau181), neurodegeneration (neurofilament light chain, NfL), and glial activation (glial fibrillary acidic protein, GFAP)., Methods: dMRI data were obtained in 111 individuals with asthma, ranging in disease severity from mild to severe, and 135 healthy controls. Regression analyses were used to test the relationships between asthma severity and neuroimaging measures, as well as AD pathology, neurodegeneration, and glial activation, indexed by plasma p-Tau181, NfL, and GFAP, respectively. Additional relationships were tested with cognitive function., Results: Asthma participants had widespread and large-magnitude differences in several dMRI metrics, which were indicative of neuroinflammation and neurodegeneration, and which were robustly associated with GFAP and, to a lesser extent, NfL. The AD biomarker p-Tau181 was only minimally associated with neuroimaging outcomes. Further, asthma severity was associated with deleterious changes in neuroimaging outcomes, which in turn were associated with slower processing speed, a test of cognitive performance., Conclusions: Asthma, particularly when severe, is associated with characteristics of neuroinflammation and neurodegeneration, and may be a potential risk factor for neural injury and cognitive dysfunction. There is a need to determine how asthma may affect brain health and whether treatment directed toward characteristics of asthma associated with these risks can mitigate these effects., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8 + tissue-resident memory (Trm) T cells. Here, we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos -/- CD8 + T cells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS deficiency or ICOS-L blockade compromised establishment of CD8 + Trm cells but not their maintenance. ICOS ligation during CD8 + T cell priming did not determine Trm induction; rather, effector CD8 + T cells showed reduced Trm differentiation after seeding into Icosl -/- mice. Icos YF/YF CD8 + T cells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos -/- Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8 + T cell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Psychological stress, an important contributor to asthma morbidity, potentiates the immune response to allergen, but the brain mechanisms mediating this response are not fully understood. The amygdala is likely to play an important role, given its sensitivity to threat and connectivity with descending immune modulatory pathways. In this study, we recruited thirty asthmatic participants and examined glucose metabolism in the amygdala, using [F-18]fluorodeoxyglucose positron emission tomography, during a laboratory stressor. Stress hormone and airway inflammatory measurements were also acquired. Results showed that activity in the amygdala was significantly increased during the stressor, compared to a matched control task (p < .05 corrected). Moreover, the increase in amygdala activity was associated with a greater increase in sputum IL-1R1 mRNA and alpha amylase response (p < .05 corrected), which were also positively correlated (p = .01). These findings suggest that heightened amygdala reactivity may contribute to asthma morbidity via descending proinflammatory sympathetic signaling pathways., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rationale: Androgens are potentially beneficial in asthma, but AR (androgen receptor) has not been studied in human airways. Objectives: To measure whether AR and its ligands are associated with human asthma outcomes. Methods: We compared the effects of AR expression on lung function, symptom scores, and fractional exhaled nitric oxide (Fe NO ) in adults enrolled in SARP (Severe Asthma Research Program). The impact of sex and of androgens on asthma outcomes was also evaluated in the SARP with validation studies in the Cleveland Clinic Health System and the NHANES (U.S. National Health and Nutrition Examination Survey). Measurements and Main Results: In SARP ( n  = 128), AR gene expression from bronchoscopic epithelial brushings was positively associated with both FEV 1 /FVC ratio ( R 2  = 0.135, P  = 0.0002) and the total Asthma Quality of Life Questionnaire score ( R 2  = 0.056, P  = 0.016) and was negatively associated with Fe NO ( R 2  = 0.178, P  = 9.8 × 10 -6 ) and NOS2 (nitric oxide synthase gene) expression ( R 2  = 0.281, P  = 1.2 × 10 -10 ). In SARP ( n  = 1,659), the Cleveland Clinic Health System ( n  = 32,527), and the NHANES ( n  = 2,629), women had more asthma exacerbations and emergency department visits than men. The levels of the AR ligand precursor dehydroepiandrosterone sulfate correlated positively with the FEV 1 in both women and men. Conclusions: Higher bronchial AR expression and higher androgen levels are associated with better lung function, fewer symptoms, and a lower Fe NO in human asthma. The role of androgens should be considered in asthma management.

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)